Chloroform (CHCL3) a hepatotoxin, has previously been shown to be oxidatively dechlorinated to phosgene (COCl2) by cytochrome P-450 in rat liver microsomes. We have further characterized the metabolism of CHCl3 in rat liver microsmes by measuring the covalent binding of (14C) CHCl3 and (3H) CHCl3 to microsomal protein as well as the fmation of chloride ion, COCl2 and CO2 under a variety of conditions. The results further confirm that CHCl3 is oxidatively dechlorinated to COCl2 by liver microsomal. In addition, a form of cytochrome P-450 that is induced by phenobarbital appears to catalyze this reaction. Once formed, COCl2 either covalently binds to microsomal protein with the release of chloride or undergoes hydrolysis to form CO2 and chloride. CHCl3 does not appear to be activated by reductive dechlorination to CHCl2 radical because the (3H) label did not bind to microsomal protein. These findings not only define more clearly the mechanism of metabolic activation of CHCl3, but also should serve as a model for determining the pathways biotransformation of other halocarbon environmental chemicals and drugs.